Microsatellites are short DNA motifs (1-10 base pairs), which occur as tandem repeats at numerous loci throughout the genome.
The microsatellite instability (MSI) phenotype is defined as the presence in tumour DNA of alternative sized microsatellites that are not seen in the corresponding germline DNA (AALTONEN et al., Science, 260(5109), 812-816, 1993; IONOV et al., Nature, 363(6429), 558-561, 1993; THIBODEAU et al., Science, 260(5109), 816-819, 1993; IACOPETTA et al., Hum. Mutat., 12(5), 355-360, 1998).
The MSI phenotype is a characteristic of the hereditary non-polyposis colorectal cancer (HNPCC) syndrome, wherein it can be detected in more than 90% of all HNPCC tumours (LIU et al., Nature Med., 2, 169-174, 1996); it also occurs in approximately 15% of sporadic colon and gastric tumours. It has also been detected in other tumours, such as pancreatic carcinomas (HAN et al., Cancer Res., 53, 5087-5089, 1993), prostate carcinomas (GAO et al., Oncogene, 9, 2999-3003, 1994), carcinomas of the endometrium (RISINGER et al., Cancer Res., 53, 5100-5103, 1993; PELTOMAKI et al., Cancer Res., 53, 5853-5855, 1993).
MSI reflects an underlying mismatch repair (MMR) defect that fails to recognize errors introduced during the replication of microsatellite sequences. In the familial cancer syndrome HNPCC, the MSI phenotype is caused by germline mutations in the mismatch repair (MMR) genes hMSH2, HMLH1 and less frequently in hPMS1, hPMS2 and hMSH6 (KINZLER et al., Cell, 87, 159-170, 1996). In sporadic cancers it is often caused by methylation of the hMLH1 promoter leading to the transcriptional silencing of this gene (HERMAN et al., Proc. Natl. Acad. Sci. USA, 95(12), 6870-6875, 1998).
MSI colonic and gastric tumours have distinctive molecular and clinicopathological profiles and are often associated with favourable prognosis (LOTHE et al., Cancer Res., 53, 5849-5852, 1993; KIM et al., Am. J. Pathol., 145, 148-156, 1994; OLIVEIRA et al., Am. J. Pathol., 153, 1211-1219, 1998). There is also evidence to suggest that colorectal cancer patients with MSI tumours show good survival benefit from 5FU-based chemotherapy (ELSALEH et al., The Lancet, 355, 1745-1750, 2000; LIANG et al., Int. J. Cancer, 101, 519-525, 2002) and therefore MSI might be a useful molecular predictive marker for response to this type of adjuvant therapy. Routine analysis of MSI status also has clinical application for assisting in the diagnosis of suspected HNPCC cases (AALTONEN et al., N. Eng. J. Med., 338, 1481-1487, 1998). Indeed, tumours from HNPCC patients lack phenotypic features that readily distinguish them from sporadic tumours and hence the diagnosis of this disease was historically based on family history of cancer using for example the Amsterdam criteria (VASEN et al., Dis. Colon Rectum, 34, 424-425, 1991; VASEN et al., Gastroenterology, 115, 1453-1456, 1999). Such criteria are too restrictive however and identify only a fraction of HNPCC families so that the true incidence of this disease is not known and estimates vary from 0.5 to 13%. Given that familial carriers of MMR defects have a greater than 80% risk of developing cancer, it is important to devise efficient and cost-effective ways to detect this condition. For this purpose, molecular-based laboratory approaches are now being developed that may help in establishing HNPCC diagnosis. Two methods are generally proposed: microsatellite genotyping and immunohistochemistry of the main mismatch repair proteins. The use of one or the other, or both of these methods is still a matter of debate, based on their relative efficiency, specificity and cost (LINDOR et al., J. Clin. Oncol., 20, 1043-1048, 2002; WAHLBERG et al., Cancer Res., 62, 3485-3492, 2002; TERDIMAN et al., Gastroenterology, 120, 21-30, 2001; LOUKOLA et al., Cancer Res., 61, 4545-4549, 2001). It appears so far that microsatellite genotyping has a higher sensitivity than IHC, but is more expensive and more difficult to set up in routine laboratories. It is thus important to develop simple and accurate methods to determine MSI tumours for predisposition and prognostic diagnosis informations.
Numerous different microsatellites have been studied by investigators with the aim of identifying MSI tumours.
Depending on the type and number of microsatellites analysed, widely variable results for the frequency of MSI in different tumour types have been published (PERUCHO, Cancer Res., 59(1), 249-256, 1999).
The use of a BAT-25 and BAT-26 marker combination has been proposed for the detection of MSI (ZHOU et al., Genes, Chromosomes & Cancer, 21(2), 101-107, 1998; HOANG et al., Cancer Res., 57(2), 300-303, 1997).
The BAT-25 and BAT-26 are mononucleotide repeats respectively located in intron 16 of c-kit and intron 5 of hMSH2. These two repeats are quasimonomorphic in Caucasian populations (HOANG et al., Cancer Res., 57(2), 300-303, 1997; ZHOU et al., Oncogene, 15(14), 1713-1718, 1997). This property allows ready classification of the large allelic size variations seen in MSI tumour DNA as being due to somatic alteration. In the large majority of tumours, analysis of BAT-25 and BAT-26 is sufficient to establish their MSI status without reference to the germline DNA (ZHOU et al., Genes, Chromosomes & Cancer, 21(2), 101-107, 1998).
However, alternative sized BAT-25 and BAT-26 alleles have been identified in 18.4 and 12.6%, respectively, of African Americans (PYATT et al., Am. J. Pathol., 155(2), 349-353, 1999; SAMOWITZ et al., Am. J. Pathol., 154(6), 1637-1641, 1999). Thus, analysis of additional repeats may be needed in order to avoid the occasional false positive result arising from these germline polymorphisms.
Accordingly it has been proposed to complete the analysis of these mononucleotide repeats by an additional analysis of dinucleotide repeats in both the tumour and germline DNA.
For instance, U.S. Pat. No. 6,150,100 proposes the use of 2 mononucleotide repeats selected from BAT25, BAT26 and BAT40, associated with 2 or 3 dinucleotide repeats selected from APC, Mfd15, D2S123, and D18S69, and optionally with the pentanucleotide repeat TP53Alu. Preferred combinations of microsatellite markers disclosed in U.S. Pat. No. 6,150,100 are BAT25, BAT26, APC, Mfd15 and D2S123 or BAT26, BAT40, APC, Mfd15 and D2S123.
In 1997 an international consensus meeting on the detection of MSI recommended a panel of five markers for the uniform analysis of MSI status (BOLAND et al., Cancer Res., 58(22), 5248-5257, 1998). This included two mononucleotide (BAT-25 and BAT-26) and three dinucleotide (D5S346, D2S123 and D17S250) repeats. Tumours with instability at two or more of these markers were defined as being MSI-H. Tumours with instability at one marker, and without instability were defined as MSI-L and MSS respectively. MSI-H cancers have distinct clinicopathological features from MSI-L and MSS tumours.
Some of the characteristics of dinucleotide repeats make their use as markers of the MSI status somewhat problematical. The dinucleotide repeats in the above panels generally show instability in only 60-80% of MSI-H tumours (SUTTER et al., Mol. Cell Probes, 13(2), 157-165, 1999). There is some evidence to suggest that loss of MMR and subsequent alteration of mononucleotide repeats occurs earlier in the MSI-H tumour progression pathway than the mutation of dinucleotide repeats (PERUCHO et al., Cold Spring Harb. Symp. Quant. Biol., 59, 339-348, 1994). Furthermore, some MSI cell lines with MMR deficiency caused by hMSH6 mutation do not show alteration in dinucleotide repeats (AKIYAMA et al., Cancer Res., 57(18), 3920-3923, 1997). Therefore the underlying MMR deficiency affecting mononucleotide and dinucleotide repeats may be different and the analysis of both may lead to misinterpretation of the MSI status of some tumours. In many instances the analysis of dinucleotide repeats adds no further information to the results obtained by analysis of mononucleotide repeats (DIETMAIER et al., Cancer Res., 57(21), 4749-4756, 1997; LOUKOLA et al., Cancer Res., 61(11), 4545-4549, 2001).
In addition, unlike mononucleotide repeats such as BAT-25 and BAT-26, dinucleotide repeats are highly polymorphic. Therefore, their use for the identification of MSI in tumour DNA always requires the analysis of corresponding germline DNA.
This makes the MSI screening process considerably more time-consuming and expensive, as well as introducing potential errors due to mixing of germline and tumour DNA samples. Moreover, the interpretation of size alterations in these dinucleotide repeats is difficult and can lead to misclassification (PERUCHO, Cancer Res., 59(1), 249-256, 1999). Finally, in many situations germline DNA from cancer patients is not readily available.
For all these reasons, the methods using BAT-26 and BAT-25 either alone, or in combination with dinucleotide repeats are not completely satisfactory for the accurate determination of MSI status in human tumours and there is an urgent need for improvement.
Thus, multiple, quasimonomorphic mononucleotide repeats are needed for the accurate diagnosis of MSI tumours.